The expression of proteins is a fundamental process in living cells. All information required for protein expression is provided by a single nucleic acid. This nucleic acid not only contains the information of the protein's amino acid sequence, it also provides the regulatory information required (e.g. the ribosomal binding site, the start and end-signals for transcription, splice signals, enhancer elements, etc.) including a promoter/promoter sequence.
A promoter is a nucleic acid that regulates the amount of transcription of a nucleic acid, e.g. encoding a polypeptide, to which it is operably linked, into pre-mRNA. It is a transcription control element, which is located around the RNA polymerase initiation site at the 5′-end of an operably linked coding sequence. From analysis of the SV40 early promoter it is known that recognition/binding sites for transcription activators are contained in promoters in segments consisting of 7-20 basepairs. One segment is the start site for RNA synthesis, e.g. the well known TATA-box. Other segments, located approximately 30-110 basepairs 5′, i.e. upstream, to the start site for RNA synthesis, are defining the frequency of transcription initiation. A promoter at least requires one segment that initiates RNA synthesis at a specific site and in a defined direction, i.e. in 5′ to 3′ direction.
Known promoters are the lac-lpp, the ara-, the lac-, the tac-, the trc-, the trp-, the phoA-, the PBAD-, the λPL-, the lpp-, and the T7-promoter. The SV40 promoter is a nucleic acid sequence derived from the genome of Simian (vacuolating) Virus 40. For the recombinant production of a heterologous polypeptide in a eukaryotic or prokaryotic cell normally one or more expression plasmids are introduced into the cell. The expression plasmid(s) comprises an expression cassette for the expression of a heterologous polypeptide and also an expression cassette for the expression of a selectable marker, which is required for the selection of transfected cells expressing the heterologous polypeptide. The synthesis of the heterologous polypeptide and of the selectable marker both requires a fraction of the cell's expression machinery's capacity.
As it is the aim to produce predominantly the heterologous polypeptide most of the available capacity of the cell's expression machinery should be allocated to the expression of the nucleic acid encoding the heterologous polypeptide. Only a minor amount should be used for the expression of the selectable marker. This allocation of expression capacity is done via the strength of the corresponding promoters. The stronger a promoter is the more of the operably linked nucleic acid is transcribed and thus translated. Therefore, it exists a need for promoters with adjustable or reducible promoter strength.
Taylor, W. E., et al. (Endocrinol. 137 (1996) 5407-5414) report human stem cell factor promoter deletion variants. In US patent application US 2007/0092968 novel hTMC promoter and vectors for the tumor-selective and high-efficient expression of cancer therapeutic genes is reported. Fromm et al. (J. Mal. Appl. Gen. 1 (1982) 457-481 and ibid 2 (1983) 127-135) report deletion mapping and deletion mutants of SV-40 early region promoter. Chitinase chitin-binding fragments are reported in U.S. Pat. No. 6,399,571. WO 99/62927 reports connective tissue growth factor-4.